Integrins mediate cell adhesion and signal transduction events that regulate many cell behaviors, including proliferation and migration. The long-term goal of this project is to understand how integrin function is modulated during differentiation. Our work to date has focused predominantly on the role of phosphorylation. The work performed under the previous granting pedod definitively supports our hypothesis that beta1 function is modulated by phosphorylation of a sedne residue in its cytoplasmic domain. Aim 1 proposes to determine how altering residues at the serine site effects interactions between beta1 and a number of structural and signaling proteins. Recent evidence suggests that interactions between vinculin and the Arp2/3 complex are critical for cell migration, and Aim 2 will investigate this migratory mechanism in F9 outgrowth cultures. Integrin function can also be modulated by lateral associations with other membrane proteins such as tetraspanins. We previously demonstrated a role for interactions between alpha6beta1 and CD9 in parietal endoderm outgrowth. Studies are proposed under Aim 3 to further explore this area. Specific Aim 1: To determine the role of serine 785 in the interaction of the beta1 cytoplasmic domain with cytoplasmic proteins and in integrin-mediated downstream signaling. We will examine the ability of 131carrying various amino acids at this site to associate with structural or signaling proteins previously identified to interact with the cytoplasmic domain using FLAG-tagged beta1versions for use in coimmunoprecipitation analysis. Specific Aim 2: To examine focal complex/adhesi0n distribution and the role of Arp2/3 complexvinculin interactions in F9-derived parietal endoderm outgrowth. We will determine the distribution, relative to the lamellipodia leading edge, of a variety of adhesion components and determine if the recently identified interaction of vinculin with the Arp2/3 complex plays a role in this outgrowth. Experiments will include the use of vinculin-deficient F9 cells. Specific Aim 3: To determine how CD9- alpha6beta1 interaction promotes parietal endoderm migration. We will determine if the CD9- (6(1 interaction promotes formation of a recently identified activated conformation of CD9 and if this activated form of CD9 is required for cell adhesion, spreading, and motility.